Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

Purification and antigenic analysis of flagella of Campylobacter jejuni

The flagella of Campylobacter jejuni strain FUM158432 were purified and a flagellin preparation consisting of only a single peptide of 63,000 daltons was obtained. The peptide of 92,000 daltons usually associated with a flagellar preparation was shown to be a peptide derived from the hook region. Antiserum was prepared by immunizing a rabbit with the flagellin preparation. The reaction of the antiserum was found to be highly specific for the flagellar filament by immunoelectron microscopy and for flagellin peptide by the immunoblotting method. Seventeen of 23 clinically isolated strains of C. jejuni reacted with this antiserum but the other six strains did not, indicating the existence of antigenic variation of the flagella of C. jejuni. The flagella of a few strains of C. coli also reacted with this antiserum.     

Identification and localization of flagellins FlaA and FlaB3 within flagella of Methanococcus voltae

Methanococcus voltae possesses four flagellin genes, two of which (flaB1 and flaB2) have previously been reported to encode major components of the flagellar filament. The remaining two flagellin genes, flaA and flaB3, are transcribed at lower levels, and the corresponding proteins remained undetected prior to this work. Electron microscopy examination of flagella isolated by detergent extraction of whole cells revealed a curved, hook-like region of varying length at the end of a long filament. Enrichment of the curved region of the flagella resulted in the identification of FlaB3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal sequencing, and the localization of this flagellin to the cell-proximal portion of the flagellum was confirmed through immunoblotting and immunoelectron microscopy with FlaB3-specific antibodies, indicating that FlaB3 likely composes the curved portion of the flagella. This could represent a unique case of a flagellin performing the role of the bacterial hook protein. FlaA-specific antibodies were used in immunoblotting to determine that FlaA is found throughout the flagellar filament. M. voltae cells were transformed with a modified flaA gene containing a hemagglutinin (HA) tag introduced into the variable region. Transformants that had replaced the wild-type copy of the flaA gene with the HA-tagged version incorporated the HA-tagged version of FlaA into flagella which appeared normal by electron microscopy.     

Caulobacter flagellar organelle: synthesis, compartmentation, and assembly.

In Caulobacter crescentus biogenesis of the flagellar organelle occurs during one stage of its complex life cycle. Thus in synchronous cultures it is possible to assay the sequential synthesis and assembly of the flagellum and hook in vivo with a combination of biochemical and radioimmunological techniques. The periodicity of synthesis and the subcellular compartmentation of the basal hook and filament subunits were determined by radioimmune assay procedures. Unassembled 27,000-dalton (27K) flagellin was preferentially located in isolated membrane fractions, whereas the 25K flagellin was distributed between the membrane and cytoplasm. The synthesis of hook began before that of flagellin, although appreciable overlap of the two processes occurred. Initiation of filament assembly coincided with the association of newly synthesized hook and flagellin subunits. Caulobacter flagella are unusual in that they contain two different flagellin subunits. Data are presented which suggest that the ratio of the two flagellin subunits changes along the length of the filament. Only the newly synthesized 25K flagellin subunit is detected in filaments assembled during the swarmer cell stage. By monitoring the appearance of flagellar hooks in the culture medium, the time at which flagella are released was determined.     

Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.     

Formation of bacterial flagella. I. Demonstration of a functional flagellin pool in spirillum serpens and bacillus subtilis

Martinez, R. J. (University of California, Los Angeles), and E. Z. Gordee. Formation of bacterial flagella. I. Demonstration of a functional flagellin pool in Spirillum serpens and Bacillus subtilis. J. Bacteriol. 91:870-875. 1966-Exponentially growing cultures of Spirillum serpens and Bacillus subtilis regained motility and flagella within one generation after mechanical deflagellation. Regeneration of flagella occurred in both cultures in the presence of chloramphenicol at concentrations shown to inhibit flagellin synthesis. Cells labeled with C(14)-amino acids regenerated radioactive flagella in the presence of chloramphenicol. A conditional mutant of S. serpens (T-45) was isolated. This strain did not produce flagella when grown at 45 C, but formed the organelles upon temperature shift to 30 C, even in the presence of chloramphenicol. A reduction of intracellular antibody-precipitable flagellin counts in labeled S. serpens T-45 occurred concomitant with the generation of flagella at 30 C. The data suggest that the flagella of S. serpens and B. subtilis are formed from a pool of intracellular flagellin proteins.     

Synthesis and Structure of Caulobacter crescentus Flagella

During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.     

Chemistry of axial filaments of Treponema zuezerae

Highly purified axial filaments have been prepared from the spirochete Treponema zuelzerae, which possess a fine structure similar to the "beaded" form of bacterial flagella. The preparations consist largely of protein but also contain small amounts of hexose (less than 1%). The buoyant density of these filaments is 1.29 g/cm(3). At pH 4.3, in the presence of 4 m urea and 10(-3)m ethylenediaminetetraacetic acid, filament protein migrates as a single band in acrylamide gel electrophoresis. Filaments dissociate to subunits in acid, alkali, urea, guanidine or with heating, indicating that these subunits are not covalently bonded in the organized structure. This is consistent with amino acid analysis which reveals that, like bacterial flagella, the filaments are completely lacking in half-cystine. Sedimentation equilibrium measurements on dissociated axial filaments in 6 m guanidine show that the subunits are homogeneous with respect to molecular weight. A weight-average molecular weight of 37,000 +/- 1,600 daltons is obtained from these measurements. The amino acid composition of axial filaments is similar to that of various types of flagellin molecules, but the filament protein is somewhat richer in tyrosine, phenylalanine, and proline than flagellin. Tryptic peptide maps of axial filaments are consistent with the amino acid composition calculated for a molecular weight of 37,000 daltons. No amino terminal end group could be detected by the dansyl chloride method, suggesting that this end group might be blocked in the axial filament protein. The results obtained show that the axial filaments of T. zuelzerae are similar chemically to bacterial flagella and suggest that they are composed of aggregates of a single species of protein subunit.     

CHLAMYDOMONAS FLAGELLA : I. Isolation and Electrophoretic Analysis of Microtubules, Matrix, Membranes, and Mastigonemes

Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ~ 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively     

Multicomponent nature of Halobacterium salinarum flagella

Filaments of the flagellum of the halophilic archaeon Halobacterium salinarum consist of five flagellins: A1, A2, B1, B2, and B3, which are encoded by five genes localized in tandem in twoflgA and flgB operons. While the role of flagellins A1 and A2 has been determined, the role of the proteins, B operon products, is still unclear. A mutant strain of H. salinarum with deleted A and B flagellin genes (deltaflgAdeltaflgB) has been obtained for the first time. This strain has been used to create and analyze the strains carrying only individual B1 or B3 flagellin genes. Cells of the deltaflgAdeltaflgB strain were shown to have short filamentous formations, 7-8 nm thick, which we have named as X-filaments. It has been shown that X-filaments consist of a protein immunologically related to flagellins A and B. Expression of the B1 and B3 genes is suppressed in the absence of A1, A2, and B2. It has been shown that flagellins B1 and B3 cannot be substituted for flagellin B2 upon the formation of a curved hook-like structure, which serves as a connecting element between the flagellar filament and the motor axis. The multicomponent nature of flagella is discussed in the light of their possible involvement in other cell processes besides providing motility.     

Localization and stoichiometry of hook-associated proteins within Salmonella typhimurium flagella.

The localization of hook-associated proteins (HAP1, HAP2, and HAP3) in Salmonella typhimurium flagella was studied by using specific antibodies together with a second antibody conjugated with colloidal gold. HAP1 and HAP3 were localized at the hook-filament junction, as has been suggested previously. HAP2, however, was localized at the filament tip. This finding supports the idea that HAP2 acts to induce polymerization of endogenous flagellin at the filament tip, and HAP1 and HAP3 are junction proteins to connect hook with filament. Analysis of the protein composition of short flagella from a mutant indicated that a single flagellum contains about 10 to 20 HAP1, 10 to 20 HAP2, and 10 to 40 HAP3 molecules.     

Recent advances in the structure and assembly of the archaeal flagellum

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.     

Various mechanisms of flagella helicity formation in haloarchaea

The genome of a halophilic archaeon Haloarcula marismortui carries two flagellin genes, flaA2 and flaB. Previously, we demonstrated that the helical flagellar filaments of H. marismortui were composed primarily of flagellin FlaB molecules, while the other flagellin (FlaA2) was present in minor amounts. Mutant H. marismortui strains with either flagellin gene inactivated were obtained. It was shown that inactivation of the flaA2 gene did not lead to changes in cell motility and helicity of the filaments, while the cells with inactivated flaB lost their motility and flagella synthesis was stopped. Two FlaB flagellin forms having different sensitivities to proteolysis were found in the flagellar filament structure. It is speculated that these flagellin forms may ensure the helical filament formation. Moreover, the flagella of a psychrotrophic haloarchaeon Halorubrum lacusprofundi were isolated and characterized for the first time. H. lacusprofundi filaments were helical and exhibited morphological polymorphism, although the genome contained a single flagellin gene. These results suggest that the mechanisms of flagellar helicity may differ in different halophilic archaea, and sometimes the presence of two flagellin genes, in contrast to Halobacterium salinarum, is not necessary for the formation of a functional helical flagellum.     

The archaeabacterial flagellar filament: a bacterial propeller with a pilus-like structure

Common prokaryotic motility modes are swimming by means of rotating internal or external flagellar filaments or gliding by means of retracting pili. The archaeabacterial flagellar filament differs significantly from the eubacterial flagellum: (1) Its diameter is 10-14 nm, compared to 18-24 nm for eubacterial flagellar filaments. (2) It has 3.3 subunits/turn of a 1.9 nm pitch left-handed helix compared to 5.5 subunits/turn of a 2.6 nm pitch right-handed helix for plain eubacterial flagellar filaments. (3) The archaeabacterial filament is glycosylated, which is uncommon in eubacterial flagella and is believed to be one of the key elements for stabilizing proteins under extreme conditions. (4) The amino acid composition of archaeabacterial flagellin, although highly conserved within the group, seems unrelated to the highly conserved eubacterial flagellins. On the other hand, the archaeabacterial flagellar filament shares some fundamental properties with type IV pili: (1) The hydrophobic N termini are largely homologous with the oligomerization domain of pilin. (2) The flagellin monomers follow a different mode of transport and assembly. They are synthesized as pre-flagellin and have a cleavable signal peptide, like pre-pilin and unlike eubacterial flagellin. (3) The archaeabacterial flagellin, like pilin, is glycosylated. (4) The filament lacks a central channel, consistent with polymerization occurring at the cell-proximal end. (5) The diameter of type IV pili, 6-9 nm, is closer to that of the archaeabacterial filament, 10-14 nm. A large body of data on the biochemistry and molecular biology of archaeabacterial flagella has accumulated in recent years. However, their structure and symmetry is only beginning to unfold. Here, we review the structure of the archaeabacterial flagellar filament in reference to the structures of type IV pili and eubacterial flagellar filaments, with which it shares structural and functional similarities, correspondingly.     

Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei.

In high (45 mM)-phosphate medium, Methanospirillum hungatei strains GP1 and JF1 grew as very long, nonmotile chains of cells that did not possess flagella. However, growth in lower (3 or 30 mM)-phosphate medium resulted in the production of mostly single cells and short chains that were motile by means of two polar tufts of flagella, which transected the multilayered terminal plug of the cell. Electron microscopy of negatively stained whole mounts revealed a flagellar filament diameter of approximately 10 nm. Flagellar filaments were isolated from either culture fluid or concentrated cell suspensions that were subjected to shearing. Flagellar filaments were sensitive to treatment with both Triton X-100 and Triton X-114 at concentrations as low as 0.1% (vol/vol). The filaments of both strains were composed of two flagellins of Mr 24,000 and 25,000. However, variations in trace element composition of the medium resulted in the production of a third flagellin in strain JF1. This additional flagellin appeared as a ladderlike smear on sodium dodecyl sulfate-polyacylamide gels with a center of intensity of Mr 35,000 and cross-reacted with antisera produced from filaments containing only the Mr-24,000 and -25,000 flagellins. On sodium dodecyl sulfate-polyacrylamide gels, all flagellins stained by the thymol-sulfuric acid and Alcian blue methods, suggesting that they were glycosylated. This was further supported by chemical deglycosylation of the strain JF1 flagellins, which resulted in a reduction in their apparent molecular weight on sodium dodecyl sulfate-polyacylamide gels. Heterologous reactions to sera raised against the flagella from each strain were limited to the Mr-24,000 flagellins.     

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.     

Chromosomal DNA from both flagellate and non-flagellate Bordetella species contains sequences homologous to the Salmonella H1 flagellin gene

Abstract The genus Bordetella contains four species: two are non-motile, the human pathogens B. pertussis and B. parapertussis ; and two are motile, the broad host-range mammalian pathogen B. bronchiseptica , and the avian pathogen B. avium . The motility of the latter two species is due to peritrichous flagella. Here we show that strains of all four species contain DNA sequences homologous to flagellin genes. Two types of gene probe were hybridised to Bordetella chromosomal DNA in Southern blots: the structural gene for H1 flagellin of Salmonella typhimurium and an oligonucleotide derived from the conserved N-terminal amino acid sequences of various flagellin proteins. Cla I-digested DNA from all four Bordetella species hybridised with both probes in Southern blots, although each species gave a characteristic pattern of hybridisation. This indicates that the non-motile B. pertussis and B. parapertussis species contain non-expressed flagellin genes.     

Role of the 25-, 27-, and 29-kilodalton flagellins in Caulobacter crescentus cell motility: method for construction of deletion and Tn5 insertion mutants by gene replacement.

Caulobacter crescentus incorporates two distinct, but related proteins into the polar flagellar filament: a 27-kilodalton (kDa) flagellin is assembled proximal to the hook and a 25-kDa flagellin forms the distal end of the filament. These two proteins and a third, related flagellin protein of 29 kDa are encoded by three tandem genes (alpha-flagellin cluster) in the flaEY gene cluster (S.A. Minnich and A. Newton, Proc. Natl. Acad. Sci. USA 84: 1142-1146, 1987). Since point mutations in flagellin genes had not been isolated their requirement for flagellum function and fla gene expression was not known. To address these questions, we developed a gene replacement protocol that uses cloned flagellin genes mutagenized by either Tn5 transposons in vivo or the replacement of specific DNA fragments in vitro by the antibiotic resistance omega cassette. Analysis of gene replacement mutants constructed by this procedure led to several conclusions. (i) Mutations in any of the three flagellin genes do not cause complete loss of motility. (ii) Tn5 insertions in the 27-kDa flagellin gene and a deletion mutant of this gene do not synthesize the 27-kDa flagellin, but they do synthesize wild-type levels of the 25-kDa flagellin, which implies that the 27-kDa flagellin is not required for expression and assembly of the 25-kDa flagellin; these mutants show slightly impaired motility on swarm plates. (iii) Mutant PC7810, which is deleted for the three flagellin genes in the flaEY cluster, does not synthesize the 27- or 29-kDa flagellin, and it is significantly more impaired for motility on swarm plates than mutants with defects in only the 27-kDa flagellin gene. The synthesis of essentially normal levels of 25-kDa flagellin by strain PC7810 confirms that additional copies of the 25-kDa flagellin map outside the flaEY cluster (beta-flagellin cluster) and that these flagellin genes are active. Thus, while the 29- and 27-kDa flagellins are not absolutely essential for motility in C. crescentus, their assembly into the flagellar structure is necessary for normal flagellar function.